Prostate cancer is the most frequently diagnosed malignancy in men and androgen ablation therapy is the main course of treatment. In many cases the disease progresses to an androgen-resistant state, for which there are few viable treatment options. Elucidation of the molecular steps of this process may identify novel points of therapeutic intervention. We have recently determined that the inhibitor of DNA binding, Id1, a member of the helix-loop-helix (HLH) family of transcriptional regulators, is downregulated in androgen-dependent, less aggressive LNCaP human prostate cells but expressed in their androgen-independent counterparts. It is also expressed in androgen-independent DU145 and PC3 cells. Moreover, Id1 is overexpressed in human prostate cancer. This supports the hypothesis that in aggressive prostate cancer cells Id1 complexes various helix-loop-helix (bHLH) binding partners to alter transcriptional activity, with an end result of promoting increased malignancy and facilitating transition to androgen independence. The proposed work addresses in detail the functional role of Id1, its E-protein binding partners, and other bHLH factors in prostate cancer malignancy with the following specific aims: 1.) The expression pattern and regulation bHLH factors, their interacting proteins and target genes in in vitro and in vivo models of prostate malignancy. 2.) By altering the levels of bHLH factors using a tetracycline-regulated expression system, the impact of these proteins on prostate cell malignancy will be determined by in vitro and in vivo assays. Overexpression of Id1 is predicted to increase malignancy while ectopic expression of E12 by alleviating the tumorigenic effects of Id1, is predicted to decrease malignancy. 3.) Pathways of Id1 and E-protein bHLH protein action in prostate will be identified by yeast two hybrid screening for Id1 and E12 binding partners and with suppressive subtractive hybridization and array-based analysis of Id1 and E-protein target genes. Id genes and bHLH factors may be suitable targets for development of new treatments for advanced prostate cancer.